The present invention generally relates to a group of fluorescent peptides for detecting the enzymatic activity of botulinum toxin type A.
Seven immunologically distinct botulinum neurotoxins, types A, B, C, D, E, F, and G are produced by the anaerobic bacterium Clostridium botulinum. Synthesized as a single 150 kD polypeptide chain in the bacteria, these neurotoxins are subsequently cleaved to produce two chains, a heavy chain and a light chain, which are linked by a disulfide bond. For each toxin, the 50 kD light (L) chain is a zinc-dependent protease, which cleaves a single target protein essential for synaptic vesicle membrane fusion during neurotransmission. Cleavage of the target protein inhibits neurotransmitter release among neurons, which leads to muscular paralysis [Bigalke and Shoer, (1999) In:Handbook of Experimental Pharmacology, Vol. 145 Bacterial Protein Toxins (K. Aktories and I. Just, Eds.) pp.407-443, Springer-Verlag, Berlin]. All references cited herein supra and infra are hereby incorporated in their entirety by reference thereto.
Clinically, there are three forms of this neuroparalytic disease: food borne, infant and wound botulism. As little as 30-100 ng of the neurotoxin is potentially lethal to humans. Thus, detection of these toxins requires a highly sensitive and reliable assay. In addition, botulinum neurotoxins are increasing useful clinically as an active ingredient in therapeutic agents. Again, sensitive and accurate potency assays are essential to assure toxin quantity, activity, and safety in any given therapeutic formulation. Currently the mouse bioassay is the xe2x80x9cgold standardxe2x80x9d for the measurement and standardization of these neurotoxins. While this in vivo assay has the prerequisite sensitivity with a detection limit of 10-20 pg neurotoxin for the most active toxin serotype, there are a number of major drawbacks. 1) The method requires the use of a large number of animals for initial detection for typing by neutralization tests and for toxin quantitation. 2) The method is costly largely due to the expense incurred for the maintenance of animal facilities. 3) The method is slow and taking up to 4 days for completion. 4) The method lacks specificity and is not reliable if the sample contains other lethal substances. 5) The method is performed by a limited number of laboratories.
Considerable efforts have been expended in the development of suitable alternative in vitro assays. Several laboratories have developed ELISA-type assays with sensitivities comparable to the mouse bioassay [Pearce et al (1997) Toxicon 35:1373-1412; Szilagyi et al (2000) Toxicon 38: 381-389]. However, these assays are based on immunodetection, and as such may not distinguish active from inactive toxin, a crucial limitation. Additionally, immunodetection may not reveal novel toxins constructed from the enzymatic portion of botulinum toxin and a binding domain of another protein. The discovery that all seven neurotoxin serotypes contain a zinc endoprotease and the identification of the specific protein substrates and cleavage sites recognized by each serotype, provides a potentially sensitive and direct means for monitoring the presence of toxin [Fujii et al (1992) Toxicon 30:1486-1488; Schiavo et al (1993) Trends Microbiol 1:170-174; Blasi et al (1993) Nature 365:160-163]. Investigations using synthetic substrates derived from synaptosome-associated protein (SNAP-25) for botulinum toxin type A and derived from vesicle-associated membrane protein (VAMP-2) for type B, demonstrated that cleavage products could be observed using RP-HPLC [Shone et al (1993) Eur. J. Biochem. 217:965-971; Schmidt and Bostian (1997) J. Prot. Chem. 16:19-26]. This method, however, does not allow for detection of small quantities of toxin. An in vitro endopeptidase assay has been described that is as sensitive as the mouse bioassay for detection of botulinum toxin type A [Ekong et al (1997) Microbiology 143:3337-3347]. The approach involves the use of antibodies directed to the neurotoxin-cleaved substrate. The assay is limited by the need for specific reagents and requires a multi step process consuming considerable time as is characteristic of ELISA assays.
A U.S. Pat. No. 5,965,699 (xe2x80x9c""699 patentxe2x80x9d) by Schmidt et al describes and claims a sensitive assay to detect cleavage of peptide substrates by botulinum toxin type A. The method uses fluorescamine to detect primary amines newly created by hydrolysis of the substrates. Using purified toxins, this method works well for tests, such as screening for potential inhibitors. However, if test samples involving food products or serum contain other sources of primary amines, such as contaminating proteins, the contaminating proteins may generate high background in the tests. The high background would hinder the detection of small quantities of toxin.
A classic approach for measuring the activity of proteolytic enzymes utilizes short, di-and tripeptide substrates which contain a chromophore or fluorophore moiety at the site of cleavage [Castillo et al (1979) Anal. Biochem. 99:53]. The chromophore or fluorophore is attached to the C-terminal of a short di- or tri-peptide through acylation which changes the electronic properties. Subsequent cleavage of the substrate and deacylation of the chromophore/fluorophore gives rise to a measurable spectrophotometric change proportional to the extent of hydrolysis. These assays do not require separation of the cleavage products. They are highly sensitive, generally relatively fast and simple to perform. However, because of the demanding substrate recognition requirements of the botulinum toxins, there are two formidable problems. First, if the neurotoxins require large peptide substrates for efficient cleavage, di- or tri-peptides would not be recognized by the neurotoxin. Secondly, and most importantly, hydrolysis of a given substrate only occurs for those peptides that contain appropriate amino acids spanning both sides of the cleavage site. Substitution of a chromophore in close proximity to the cleavage site, which is necessary when using this technique, would eliminate hydrolysis.
More recently, an alternative approach, based on intramolecularly quenched fluorescent substrates has been successfully utilized to characterize various peptidases [Szollosi et al (1998) Cytometry 34:159-179]. These fluorogenic substrates contain a fluorescent group at one end and a suitable chromogenic group that can quench the fluorescence at the other. Substrates may be designed to include amino acids on either side of the enzymatically hydrolyzed bond. The fluorescent signal in the uncleaved substrate is quenched by fluorescence resonance energy transfer (FRET) between the fluorophore and quencher groups. Efficiency of quenching depends on i) the distance between the donor/acceptor groups, the shorter the distance the more efficient the quenching, and ii) the extent to which the emission spectrum of the fluorophore and the absorbance spectrum of the chromophore overlap [Yaron et al (1979) Anal. Biochem. 95:228-235]. The greater the spectral overlap a given donor/acceptor pair has, the more efficient the quenching of the signal will be. Full fluorescence is restored after cleavage of the peptide. Hence, enzymatic activity can be monitored continuously by recording the increase in fluorescence intensity with time.
A similar intramolecularly quenched fluorescent substrate is described and successfully used for determining botulinum toxin type B protease activity [Anne et al (2001) Anal. Biochem. 291:253-261]. The 34 amino acid substrate described is a contact quenched peptide. The mechanism of quenching is not by FRET, but is collisional. For these contact quenched substrates, the fluorophore and quencher must be in close proximity. In this substrate, they are separated by only four amino acids. Even though the modified amino acids are quite close to the cleavage site, this peptide substrate is still recognized by the neurotoxin. A similar substrate specific for tetanus toxin, however, was not cleaved, indicating that the positioning of the fluorophore and quenching groups is crucial in constructing a suitable substrate and emphasizing that the design of useful fluorescent substrates is not trivial.
In this invention, we describe a series of FRET substrates for botulinum toxin type A (BTA). It has been shown previously that a 17-amino acid peptide from the SNAP-25 target protein is hydrolyzed as efficiently as the full substrate [Schmidt and Bostian (1995) J. Prot. Chem. 14:703-708]. It was also noted that, while reduction to a 13 amino acid substrate decreased the hydrolysis by 43%, this peptide was still a good substrate [Schmidt and Bostian (1997) ibid.]. If the FRET pairs are separated by 17 amino acids, quenching would be minimal. Thus we have designed and synthesized thirteen 13-15 amino acid peptides using three different FRET pairs, o-aminobenzoyl (Abz)/2,4 dinitrophenyl(Dnp), Abz/3-nitro-tyrosine, and (7-methoxycoumarin-4-yl)acetyl(Mca)/Dnp. One substrate containing Mca/Dnp and some of the substrates containing Abz/Dnp, were cleaved by the toxin, indicating that these FRET pairs do not interfere with binding and cleavage and that this method is feasible.
In this invention, we describe the design, construction, catalytic properties and overall evaluation of 13 new FRET peptide substrates for assessing the protease activity of botulinum toxin type A. These substrates may be used in a highly sensitive, rapid and relatively inexpensive in vitro assay for 1) detecting low levels of toxin contamination in food, clinical, and environmental samples, 2) evaluating the quantity and safety of therapeutic agents containing botulinum toxin type A, 3) monitoring the production of botulinum toxin type A by fermentation processes, 4) detecting toxin neutralizing antibodies, as well as 5) screening and characterization of a large number of toxin inhibitors, which are potential therapeutic agents.
The present invention relates to a new type of peptide substrate for the determination of the endoprotease activity of C. botulinum type A neurotoxin. It is well known that fluorescent dyes absorb light energy and emit it at a lower energy producing fluorescence. If a second chromophore is within a certain distance of the dye and absorbs light at the wavelength of the emitted light, then the absorbed light can be transferred to the second chromophore. In other words, the second compound or acceptor quenches the light emitted by the first compound or donor. The process is referred to as Fluorescence Resonance Energy Transfer (FRET). A pair of compounds which interact in this matter as a donor and acceptor is called a FRET pair. The effectiveness of this technique depends on the distance between the FRET pair as well as the amount of overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor. The efficiency of energy transfer between the donor and acceptor is inversely related to the sixth power of the distance between the two. When the FRET pair on a single peptide is separated due to cleavage of the peptide, a change in the fluorescent emission occurs. In this case, light absorbed by the donor is emitted at a longer wavelength and is detected using a fluorimeter. For a review see Wu and Brand (1994) Biochem, 218, 1-13.
The present invention describes a peptide suitable for digestion by a specific protease as well as a method using both peptides and antibodies to selectively detect the C. botulinum type A endoprotease if other proteases are present. Utilizing the known sequence of the cleaved area of SNAP 25, relatively short peptides were designed to include amino acid substitutions intended to decrease the suitability as a substrate for other proteases while maintaining its function as a substrate for botulinum type A toxin. Additionally, several FRET pairs were bound to these peptides to determine which would provide a minimum of background fluorescence and maintain the susceptibility of the peptide to digestion by C. botulinum type A endoprotease.
Historically, antibodies have been used to distinguish the seven different serotypes of C. botulinum. Monoclonal antibodies specific to different regions of C. botulinum neurotoxin type A have been described [Hallis et al (1993) In: Botulinum and Tetanus Neurotoxins (B. R. DasGupta, Ed.) pp. 433-436, Plenum Press, New York; Pless et al (2001) Infect. Immun. 69:570-578; Wu et al (2001) Appl. Environ. Microbiol. 67:3201-3207]. The peptide substrates described in this invention specifically detect the protease activity of the light chain of C. botulinum type A. Elimination of the protease activity by an antibody directed to the enzymatic portion of the neurotoxin creates a highly specific method of identification.
In addition, Botulinum neurotoxins have several properties which allow them to be seperated from a sample containing other materials including other proteases. Examples of such properties include the fact that the neurotoxin will adhere to chromatography media or precipitate as described in the classical purification schemes [DasGupta and Sathyamoorthy (1984) Toxicon 22:415-424]. Botulinum toxins may also be captured using a solid state bound antibody [Wictome et al (1999) FEMS Immunology and Medical Microbiology 24:319-323].
Therefore, it is an object of the present invention to provide substrate peptides for use in an assay for determining the enzymatic activity of botulinum type A neurotoxin. Such substrates must be carefully designed to meet the demanding requirements of the serotype A neurotoxin. Selection of an efficient donor/acceptor pair is critical for maximum quenching in the uncleaved substrate. In turn, this FRET pair must not interfere with recognition, binding, or catalysis by BTA.
It is another object of the present invention to provide a method for measuring and detecting the proteolytic activity of botulinum type A neurotoxin in a sample. This method may be used with a standard curve to quantitate the toxin. Alternatively, the method may simply be used for a qualitative determination of the presence of the toxin. This method may be applied to monitoring the production of botulinum type A toxin in fermentation. The method can also be used in evaluating the quantity and safety of therapeutic agents containing botulinum type A toxin. The specificity of the assay described in the present invention can further be increased by first enriching the concentration of neurotoxin by using chromatography media or precipitation. The neurotoxin in a sample can also be captured using a solid state bound antibody.
It is further an object of the present invention to provide a method for screening large numbers of inhibitors against botulinum type A toxin or compounds that have stimulatory characteristics to botulinum type A toxin. Inhibitors may be useful in treating patients with botulinum type A intoxication, while the stimulatory compounds may be used to enhance the activity of neurotoxin-containing drugs.